Copper appeared in biology after the great oxidation event and has become an essential cofactor for all forms of aerobic life. Its reactivity required the evolution of specific copper handling pathways involving transporters and chaperones in every compartment in the cell. We have developed Chlamydomonas as a reference organism for understanding copper homeostasis in eukaryotic cells, especially in the context of deficiency and in competition with other essential metals like zinc and iron. Previously, we enumerated the cuproproteome of Chlamydomonas, identified a key conserved copper-sensing transcription factor and detailed the copper regulon. Now, we report the discovery and biochemical characterization of a chloroplast inter-membrane space-localized copper chaperone, PCH1, which arises from an alternate splicing event that is conserved over a billion years of evolution. PCH1 can transfer Cu* with high selectivity for its cognate transporter PAA1 in the envelope membrane, but not PAA2 in the thylakoid membrane. A second major discovery is of the cuprosome, a lysosome-related compartment, which can accumulate bio-available Cu. Cu* was visualized by a chemical fluorescent sensor and validated by NanoSIMS, a state of the art physical technique. We developed heavy isotope pulse-labelling methods to demonstrate that cuprosome Cu* is bioavailable, which has broad impact in terms of handling Cu* hyperaccumulation sites resulting from genetic defects in mouse and human. In ongoing work, we are pursuing functional studies of 1) peptidylglycyl a- amidating monooxygenase, a conserved cuproenzyme in animals and humans, localized to cilia, whose biology is not yet well understood, 2) RSEP1, a thylakoid membrane-localized protease responsible for Cu recycling in Chlamydomonas, and 3) other components of the Cu regulon. Finally, we have used RNA-Seq transcriptome profiling to distinguish the zinc regulon and expand the copper regulon. During the next period, we will 1) purify the Cu*-binding ligand in the cuprosome, 2) use high throughput genetic analyses to identify mechanisms of loading and unloading the cuprosome, 3) distinguish whether zincosomes, cuprosomes and toxic metal sequestering sites are identical or specialized versions of the same compartment, 4) identify additional zinc- and copper-responsive transcription factors / sensors in Chlamydomonas via genetic screens, and 5) continue functional studies of the target genes of the copper and zinc regulons, including especially novel pioneer components, as well as candidate copper homeostasis factors involved in copper movement between compartments. RELEVANCE (See instructions): Copper and zinc are essential micronutrients for all forms of life because they function in proteins to catalyze particular chemical reactions in cells. Yet, many organisms, including humans face deficiency because of inadequate diet or genetic disease. In this project we investigate the compensatory mechanisms that occur at the level of the genome, at the level of cell structure as well as intermediary metabolism, in response to such deficiencies, using a reference organism.